Hapten-protein conjugates for use in detection of organophosphorus compounds

ABSTRACT

Novel generic antibodies are provided that are specific to groups of organophosphates, particularly to a class of organophosphate pesticides in common usage. Further provided are compounds and methods for raising these antibodies, test kits containing them and methods for their use. The antibodies targeted at low molecular weight (i.e. below MW 1000) organophosphorous pesticides methacrifos, pirimphos methyl, etrimfos, fenitrothion, chlopyrifos methyl, dichlorovos and malathion. Preferred conjugates of the invention are of the formula (RO) 2  --P(S)--Z-- Y!-Protein wherein R is lower alkyl and Z is as O, S or --NH--, Y is a spacer group and &#34;Protein&#34; indicates a protein suitable for use in hapten protein conjugates for the purpose of raising antibodies and antisera. Typical and preferred proteins are bovine serum albumin, ovalbumin from chicken egg, keyhole limpet hemocyananin and mixtures of these.

The present invention relates to novel generic antibodies that arespecific to groups of organophosphates, particularly to a class oforganophosphate pesticides in common usage. Further provided are haptencompounds and methods for using them in raising these antibodies, testkits containing them and methods for their use.

A number of immunoassays are available for the detection of variousbiologically important compounds. Such compounds include the importantorganophosphate (OP) class of pesticides (see eg. WO 91/00294). Theseimmunoassays are relatively sensitive, but are directed againstindividual OP molecules and thus several separate assays are required toeffectively screen a material for OP presence.

A group of these organophosphate pesticides that is commonly used forfoodstuff, eg. grain, protection comprises the compounds methacrifos,pirimphos methyl, fenitrothion, etrimfos, propetamphos, chlopyrifosmethyl and dichlorvos, and screening a material for the presence of anyof these potentially toxic compounds conventionally requires use ofseven separate agents. While these compounds share some chemicalfeatures with each other, including that of the OP group itself, othercommonly used pesticides are distinguished by variant OP groups. Inmalathion for example a (CH₃ O)₂ P(S)--S-- group is present as comparedto the (CH₃ O)₂ P(S)--O-- group of the compounds referred to above.

The present inventors have now provided a novel strategy for raisingantibodies such that they are specifically directed to bind with two ormore of these specified pesticidal organophosphorous compounds andfurther provide methods for using these and kits containing them, suchthat materials can be screened for presence of a member of a selectedgroup with a single antibody reagent. Still further they have providedorganic haptens and their conjugates which may be used specifically toraise these antibodies, or antisera containing them, as eithermonoclonal or polyclonal antibodies. Particularly provided areantibodies capable of specific binding with two or more, more preferablythree or more, and most preferably all of the compounds methacrifos,fenitrothion, etrimfos, propetamphos, dichlorvos and optionally one ormore of malathion, dimethoate, chlorfenvinphos, chlopyrifos methyl,tetrachlorvinphos and pirimiphos-methyl, wherein the relative bindingaffinity with each of these bound compounds is such that a singlestrength antibody reagent can be used to bind them. Preferably the OPsbound are capable of displacing percentages of bound hapten from theantibody at a concentration of 100 μg/ml within a factor of 20, morepreferably of 5, and more preferably 2 of each other. Also provided arehaptens and hapten-protein conjugates which evoke production of suchantibodies when adminstered to animals.

The concept that one generic antibody or antisera might be raised to becapable of detecting a number of organophosphates has been considered onone occasion previously by Suedi and Heesham Kiel: (1988) MilchwirtForsch 40 179-202, but this involved use of a number of model pesticidemolecules to provide a number of protein conjugates. Furthermore theantibody reagents obtained were not capable of use in reliablescreening.

In a first aspect of the present invention there is provided a haptenprotein conjugate capable of stimulating production of antibodies whichhave specific binding affinity for compounds including the chemicalmoiety I:

    (RO).sub.2 P(S)--A--                                       (I)

wherein R is lower alkyl, particularly C₁₋₄ alkyl, and A is O or S;characterised in that the antibody binds two or more, preferably threeor more, and most preferably all of the compounds methacrifos,fenitrothion, propetamphos, dichlorvos, dimethoate, malathion,chlorfenvinphos and etrimfos; preferably also binding to chlopyrifosmethyl, tetrachlorvinphos and pirimiphos-methyl.

Most preferably the conjugates evoke production of antibodies targetedat moiety (I) where R is methyl, and preferably where A is O, whereincross-reactivity with other compounds relative to those having thismoiety is low enough to enable ready identification of the targetedcompounds in a mixture by binding with a known antibody concentration.

Preferred hapten protein conjugates of the invention are of formula(II):

    (RO).sub.2 --P(S)--Z-- Y!-Protein                          (II)

wherein R is as defined for moiety (I) and Z is O, S or --NH--, Y is aspacer group and `Protein` indicates a protein suitable for use inhapten-protein conjugates for the purpose of raising antibodies andantisera. Typical and preferred proteins are bovine serum albumin,ovalbumin from chicken egg, keyhole limpet hemocyanin and mixtures ofthese. Preferably linkage to protein is by one of its amino groups.

Preferred spacer groups are format --(CH₂)_(n) --B-- wherein n is suchthat the carbon chain length is 4 to 6 and B is CO or O(CO), or thespacer is a pyridine or pyrimidine ring with a CO or O(CO) group.Optionally the spacer may have side chains, eg. as in compounds such asβ-alanine as disclosed by WO 9100294, but the most preferred conjugatesof the invention have a straight carbon chain of 4-6 eg. a1,4-butanediol spacer whereby Z is O and B is --O(CO)-- provided by wayof carbodiimide activation of the --OH end group. Examples of suitablehaptens are listed as follows:

(CH₃ O)₂ --P(S)--O--(CH₂)₄ --O--CO--NH-Protein (butandiol spacer)

(CH₃ O)₂ --P(S)--O--(CH₂)₄ --CO--NH-Protein (butandiol spacer)

(CH₃ O)₂ --P(S)--O--CH₂ --CH₂ --O--CO--NH-Protein (3 carbon spacer)

(CH₃ O)₂ --P(S)--O--CH₂ --CH₂ --CO--NH-Protein (3 carbon spacer)

(CH₃ O)₂ --P(S)--O--CH₂ --O--CO--NH-Protein (2 carbon spacer)

(CH₃ O)₂ --P(S)--O--CH₂ --CO--NH-Protein (2 carbon spacer)

(CH₃ O)₂ --P(S)--S--CH₂ --CH₂ --O--CO--NH-Protein (SH-linked spacer)

(CH₃ O)₂ --P(S)--S--CH₂ --CH₂ --CO--NH-Protein (SH-linked spacer)

(CH₃ O)₂ --P(S)--O-pyridine-O--CO--NH-Protein (pyridine spacer)

(CH₃ O)₂ --P(S)--O-pyridine-CO--NH-Protein (pyridine spacer)

(CH₃ O)₂ --P(S)--O-pyrimidine-O--C--NH-Protein (pryrimidine spacer)

(CH₃ O)₂ --P(S)--O-pyrimidine-CO--NH-Protein (pryrimidine spacer)

In a second aspect of the invention there are provided hapten compoundsof formula (III) for use in preparing compounds (II):

    (RO).sub.2 --P(S)--Z--(Y)--B--(D)                          (III)

wherein R, Z, B and Y are as described above and D is H or an activatorgroup. Typical activator groups are those such as imidazole residues ofthe type left by activation with carbodiimides, but halogen atoms mayalso used where B is CO, such as to form an acid halide, capable ofactivating reaction between the compound and protein amino groups.Particularly provided are hapten compounds of formula (IV):

    (RO).sub.2 P(S)--O--(Y)--B--(D)                            (IV)

wherein Y is a spacer of four carbon length, with or without sidechains, preferably being a --(CH₂)₄ -- moiety. Most preferably B isO--CO-- and D is an imidazole residue. Where D is H the compound is anunactivated hapten, otherwise the compound is in the activated form.

Further provided by the present invention is a method for synthesis ofthe haptens and conjugates of formula (II), (III) and (IV) wherein adialkyl halothiophosphate is reacted with a spacer group precursor toproduce the hapten. The resultant hapten is then activated with anactivator moiety to provide the activated product and that is conjugatedwith the protein to provide a protein conjugate of formula (II) in theusual manner.

The dialkyl halothiophosphate/spacer group reaction is convenientlycarried out in organic solvent phase with a base, eg. pyridine, and atlow temperature, eg between -5° and 10° C., preferably 0° and 5° C.Acetone solvent is conveniently used. The reaction mixture is preferablyshaken or stirred with the spacer group being dissolved in thesolvent/base mixture and the dialkyl halothiophosphate being addedslowly to this before reflux, that preferably being at about 60° C. fora period of several, but most preferably about two, hours. Theactivation of the hapten is conveniently carried out using acarbodiimide, eg. carbonyl diimidazole, in the known manner and thisactivated hapten is then conjugated with the selected protein.

The antibodies and antisera produced using these conjugates of theinvention can be raised by any of the methods conventionally used in theart using any of the conventional animal donors. Furthermore theantibodies so raised may be polyclonal or monoclonal; the latter beingderivable by hybridoma technology as will be well understood by thoseskilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

For OP formulae see FIGS. 1A, 1B, 1C, 2A, 2B, 2C, 2D and 2E. The presentinvention further provides test kits comprising the antibodies orantisera of the invention together with other items specific for thedetermination of the presence of the target organophosphates in amaterial. Such items may include calibration reagents containing a knownamount of target OP compound, or may take the form of antibodiesimmobilised on a substrate, eg. on microtitre well plates or the like,whereby absorbance values may be readily obtained at suitablewavelength, eg. 450 nm, to determine antibody titre, or other reagentssuch as specific buffers, blocking agents or colour developers. Typicalkit formats are as discussed in WO 9100294.

The production of antibodies and the antisera containing them, and theiruse in assays, is carried out using known methods and is exemplified inthe Examples. The haptens, conjugates, antibodies, antisera and methodsof the present invention will now be described by way of example only byreference to the following non-limiting Examples.

EXAMPLE 1 Synthesis of Generic Organophosphate Antibody GeneratingConjugate Hapten Intermediate of Formula (III) and (IV)

10 mmoles of pyridine (0.79 g) were dissolved in 5 ml acetone and thesolution kept on ice. To this was added 10 mmoles of 1,4 butanediol(0.90 g) and with constant shaking of the chilled mixture using amagnetic stirrer 10 mmoles of dimethylchlorothiophosphate (CH₃ O)₂--P(S)Cl (1.61 g) was slowly added. The reactants were refluxed at 60°C. for 2 hours and then left overnight at room temperature (Reaction A).

The final product was purified by extracting the mixture of reactantswith 3×25 ml of diethylether, washing that with 3×10 ml of H₂ O and thenevaporating it to a final volume of about 2 to 3 ml. Formation of (CH₃O)₂ P(S)--O--(CH₂)₄ --OH (mol wt. 214) was indicated on TLC (Rf=0.1 inchloroform:acetone 70:30) and confirmed on GC/MS. ##STR1##

EXAMPLE 2 Activation of Hapten

Example 1 was treated with 10 mmoles (1.62 g) of carbonyl diimidazolethus providing activated hapten solution. (NB. Where B is a carboxylgroup thionyl halide may be used to produce the corresponding acidhalide in the known manner).

EXAMPLE 3 Formation of Protein Conjugates

3 mmoles of activated hapten from Example 2, in the form of 1 ml of theethereal solution, was added very slowly--one drop every fiveminutes--to each of three protein solutions whilst constantly stirringusing a magnetic stirrer; these containing 30 μmoles of BSA orOvalbumin, or 3 nmoles of Keyhole Limpet hemocyanin respectively in 10ml phosphate buffer saline pH 7.4. Reactants were stirred slowly for 2hours, left overnight at room temperature and then at 4° C. for 72 hours(see Reaction B). The conjugate solutions were analysed for theconcentration of protein using the Coomassie blue method of Bradford:Analytical Biochem. (1976) 72:248-254. The ratio of the conjugation wasfound by the free amino group method of Habeeb as in Analytical Biochem(1966) 14:328-336 as an OP:BSA of 10 and an OP:Ovalbumin of 4.

Purification of conjugate was effected by application to Sephadex (PD10) columns, with dialysis of insoluble or sparingly soluble conjugatesagainst distilled water being carried out as a preparatory step prior toapplication. ##STR2##

EXAMPLE 4 Production of Polyclonal Antibodies to Generic Conjugate

Simone Noir half lop rabbits were housed individually and allowed 3weeks to settle in before pre-immunisation blood samples were taken andthe immunisation schedule started. Hapten-Bovine serum albumin (BSA)conjugates were first mixed with equal volumes of Freund's adjuvant sothat the final concentration was about 1 mg/ml of protein.

After testing to show the absence of native antibodies reactive with theconjugates, each rabbit was given subcutaneous injections of 1 ml of theBSA generic conjugate of Example 3 spread over 4 sites (0.25 ml persite) in complete adjuvant. Subsequent injections were given at 4, 8 and12 weeks in incomplete adjuvant. When the antibody titre started to fallthe rabbits were boosted with a further 1 ml of conjugate in Freund'sincomplete adjuvant spread over 4 sites. In the tables below rabbitsR19,20,21 were immunised with this generic conjugate, ratio OPhapten:protein of 10:1, of the invention.

Blood was taken from the marginal ear vein prior to immunisation forantibody checks and at 4 and 14 weeks after injection in order thatantisera stocks could be built up. Antisera were prepared by allowingblood to clot overnight at 4° C. and then centrifuging it to provideclear fluid. Antibodies could be isolated from this as required by useof ion exchange or affinity column chromatography with immobilisedtarget organophosphate groups in the conventional manner.

EXAMPLE 5 Indirect:Antibody Capture Enzyme Linked Immunosorbent Assay(ELISA) Demonstrating Affinity of Antisera for Moiety of Formula (I)

An ELISA was used to screen the antisera for polyclonal antibodies tothe generic hapten/conjugate of the invention using the followingprotocol. Microtitre plates (Costar) were coated with the appropriatehapten conjugate with ovalbumin by diluting that in 0.05M sodiumhydrogen carbonate/sodium carbonate buffer (pH 9.6) to give an optimumprotein concentration of 1 μg/ml previously determined by chequerboardtitration; protein found by Bradford assay. 100 μl was added to eachmicrotitre plate well and the plates placed in a moist chamberovernight, washed 3 times with 0.15 phosphate buffered saline pH7.2 with0.05% Tween 20 RTM!: PBST), and blotted dry. Blocking was carried out byadding 250 μl per well of 5% skimmed milk for 1 hour at 25° C.

After two further washings the coated microplates were stored at -20° C.until required, whereon they were washed once with PBST. Antisera werediluted (1/1000 for general screening or doubling dilutions up to1/512,000 for titration assays) in PSBT, applied to microtitre wells intriplicate in 50 μl amounts and incubated in a moist chamber at 25° C.for 1 hour (appropriate positive and negative controls wereincluded--PBST, negative rabbit serum and positive serum when it hadbeen produced). Wells were then washed 3 times in PBST and 200 μl of a1:1500 dilution of swine anti-rabbit immunoglobulin conjugated tohorseradish peroxidase (DAKO) was added to each well. Plates wereincubated once more for 1 hour at 25° C. then washed 3 times with PBST.

Tetramethylbenzidine (TMB) enzyme substrate solution was prepared byadding 400 μl of 6.0 g/l TMB in DMSO to 24.5 ml of 0.1M sodium acetatebuffer pH5.5 with 100 μl of 1% H₂ O₂ and 100 μl added to the wells. Themicroplates were incubated for 15 minutes in the dark at 25° C. beforethe reaction was stopped by adding 50 μl of 2.5M H₂ SO₄ and theabsorbance read at 450 nm against air. Results: are given below withthose of Example 6.

EXAMPLE 6 Competitive/Inhibition Assay

This assay was carried out in the same manner as that of Example 5 withthe following modifications. After plate coating the various dilutionsof antisera were incubated with various concentrations of analytesolution for 1 to 2 hours at 25° C. or overnight at 4° C. Antiserumdilutions tested were 1/1000, 1/2000, 1/5000 and 1/10000 and theconcentrations of the analyte ranged from 200 to 0.001 μg/ml. Coating ofgeneric hapten-ovalbumin was at 1 μg/ml and later other coatingstrengths were tested ranging from 400 to 12.5 ng/ml. Note: crossreactivity in all cases was checked by carrying out an indirect ELISAwhere plates were coated with the blocking agents or BSA or ovalbumin at1 μg/ml. Skimmed milk, foetal calf serum (both 5%), caesin (0.3%) orfish skin gelatin (0.5%) were selected as the blocking agent in thisregard. Use of competitive assay is preferred for determiningcross-reactivity.

Results: Sera collected at 4 and 16 weeks showed reactivity with theconjugate, and chequerboard titrations at 16 weeks (see Table 1 and 2)showed all three rabbit's sera to be strongly reactive. Optimum coatingof wells with the conjugate proved to be achieved with about 1 μg/ml,giving high absorbance with antisera and low absorbance with negatives.It should be noted that cross-reaction with spacer moiety or proteinalone may occur; skimmed milk coated, gelatin blocked plates were shownto obviate this sufficiently for successful ELISA.

Table 3 shows the results of the competitive/inhibition ELISA indicatingthat the coating levels should be less than 1 μg/ml for optimalsensitivity: eg. 400 to 0.012 μg/ml conjugate for 200 to 0.001 μg/mlanalyte detection.

                  TABLE 1                                                         ______________________________________                                        Chequerboard absorbance with values for 16 week generic BSA                   conjugate antisera (1/1000) with coating concentrations of 20 to              0.16 μg/ml OP-Ova                                                          Coating Conc.                                                                           Rabbit 19                                                                              Rabbit 20 Rabbit 21                                                                             Negative                                 ______________________________________                                        20.0      2.22     2.30      2.22    0.17                                     10.0      2.15     2.20      2.21    0.16                                     5.0       2.05     2.21      2.18    0.14                                     1.25      1.87     2.11      2.06    0.13                                     0.63      1.51     2.07      1.99    0.14                                     0.32      1.36     1.94      1.86    0.18                                     0.16      0.92     1.75      1.41    0.21                                     ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Absorbance values for 16 week generic BSA conjugate                           antisera screen and dilution when negative (absorbance                        <0.2) plate coated at 1 μg/ml with OP-Ova at 1 μg/ml.                              Absorbance Dilution when negative                                  Rabbit No. (serum 1/1000)                                                                           (<0.2)                                                  ______________________________________                                        19         2.44       1/512000                                                20         2.56       1/512000                                                21         2.56       1/512000                                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Competitive/inhibition assay for antisera from rabbits 19, 20, 21             pre-incubated with various concentrations of unconjugated                     organophosphate (OP) for 1 hour at 25° C.: 1 μg/ml OP-Ova           Conc. of OP μg/ml                                                          as preincubated                                                               with antisera                                                                              Rabbit 19   Rabbit 20                                                                              Rabbit 21                                   ______________________________________                                        50.0         1.34        1.19     1.24                                        12.5         1.36        1.31     1.02                                        3.1          1.50        1.54     1.34                                        0.4          1.66        1.81     1.45                                        0.1          1.73        1.95     1.42                                         0.01        1.83        1.95     1.52                                        Without OP   2.30        2.39     --                                          ve serum     0.13        0.13     0.15                                        ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Competitive/inhibition assay for antisera: preincubated with                  OP for 2 hours at 25° C. Plate coated with 400 μg/ml OP-Ova.        OP conc as preinc'                                                                             Dilution of Rabbit 20 antisera                               with antisera    1/1000   1/2000                                              ______________________________________                                        200.0            0.89     0.69                                                100.0            0.82     0.67                                                20.0             0.87     0.67                                                10.0             0.94     0.74                                                1.0              1.24     0.87                                                0.1              1.48     1.04                                                0.01             1.58     1.11                                                0.001            1.38     1.10                                                Without OP       2.00     1.69                                                ______________________________________                                    

EXAMPLE 7 Modified Competitive Inhibition Assay

Assay was carried out in the same manner as Example 5 with the followingmodifications. Antisera was diluted 1:2000 with 1% BSA in PBST and 100μl was incubated for 2 hours at 25° C. with 100 μl of each concentrationof the unconjugated OP--in this case the generic part--in amicrotitration plate. Concentrations of OP ranged from 200 to 0.001 μgml⁻¹.

Cross reactivity of antisera from rabbits 19, 20 and 21 by inhibitionELISA with the various organophosphorus pesticides is shown in Table 5in which concentration (μg ml⁻¹) for 50% inhibition to occur is giventogether with the diluent in which this is achieved--PBST or % methanolin PBST for figures.

                  TABLE 5                                                         ______________________________________                                        Inhibitor   Rabbit 19 Rabbit 20    Rabbit 21                                  ______________________________________                                        Hapten + spacer                                                                           +++       +++ (3.0 PBST)                                                                             +++                                        Hapten no spacer                                                                          ++        poor         ++                                         Spacer      - (none)  - (none)     - (none)                                   Fenitrothion                                                                              +         +++ (4.8 10%)                                                                              +                                          Methacrifos +         +++ (8.2 PSBT)                                                                             +                                          Propetamphos                                                                              +         +++ (36.2 10%)                                                                             ++                                         Dichlorvos  ++        ++ (91.1 PSBT)                                                                             +                                          Dimethanoate                                                                              - (none)  ++ (>150 10%)                                                                              +                                          Malathion   - (none)  +            - (none)                                   Chlorfenvinphos                                                                           - (none)  +            +                                          Etrimfos    - (none)  +            poor                                       Chlorpyrifosmethyl                                                                        poor      poor         poor                                       Tetrachlorvinphos                                                                         - (none)  poor         poor                                       Pirimiphos-methyl                                                                         - (none)  poor         - (none)                                   Glyphospate - (none)  - (none)     - (none)                                   ______________________________________                                         +++ strong                                                                    ++ medium                                                                     + weak reaction                                                               poor--slight                                                             

The relative affinities of the various organophosphates for the genericconjugate antibodies as shown by the results from Example 7 are providedbelow as derived by subtracting the amount of inhibition of binding at aconcentration of 0.1 μg/ml organophosphate from that obtained with 100μg/ml organophosphate; results are presented as % of the inhibitionobtained with hapten including spacer as the inhibitor.

                  TABLE 6                                                         ______________________________________                                        Relative inhibition of binding of generic antibodies                          to hapten conjugate as % of hapten + spacer inhibition                        Organophosphate Percentage inhibition                                         ______________________________________                                        Hapten-spacer   100.0                                                         Fenitrothion    90.1                                                          Methacrifos     89.1                                                          Propetamphos    75.9                                                          Dichlorvos      60.0                                                          Dimethoate      41.4                                                          Malathion       38.6                                                          Chlorfenvinphos 33.4                                                          Etrimfos        30.8                                                          Chlorpyrifos-methyl                                                                           29.2                                                          Tetrachlorvinphos                                                                             27.2                                                          Pirimiphos-methyl                                                                             21.1                                                          ______________________________________                                    

We claim:
 1. A hapten-protein conjugate having the formula:(CH₃ O)₂ --P(S)--O--(CH₂)₄ --O--CO--NH-Protein, or (CH₃ O)₂ --P(S)--O--(CH₂)₄ --CO--NH-Protein wherein Protein indicates a protein suitable for use in said hapten-protein conjugate for the purpose of raising antibodies, antisera or use in immunoassays.
 2. A conjugate as claimed in claim 1 wherein the protein is selected from the group consisting of bovine serum albumin, ovalbumin from chicken egg and keyhold limpet hemocyanin.
 3. A method for synthesis of said hapten protein conjugate of claim 1 comprising (a) reacting a dialkyl halothiophosphate with a spacer group to produce said hapten, (b) activating the resultant hapten with an activator moiety to provide an activated product, and (c) reacting the product of step (b) with the protein. 